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Abstract

Background

The interplay between IFN-γ, IL-17 and neutrophils during CNS inflammatory disease
is complex due to cross-regulatory factors affecting both positive and negative feedback
loops. These interactions have hindered the ability to distinguish the relative contributions
of neutrophils, Th1 and Th17 cell-derived effector molecules from secondary mediators
to tissue damage and morbidity.

Methods

Encephalitis induced by a gliatropic murine coronavirus was used as a model to assess
the direct contributions of neutrophils, IFN-γ and IL-17 to virus-induced mortality.
CNS inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed
wild-type (WT) or IFN-γ deficient (GKO) memory CD4+ T cells into infected SCID mice, coupled with antibody-mediated neutrophil depletion
and cytokine blockade.

Keywords:

Background

IL-17 and IFN-γ play diverse and often opposing functions during microbial infections,
as well as autoimmune diseases. These interactions are partially attributed to their
distinct regulation of the neutrophil response. Both IL-17A and IL-17 F signal through
the IL-17R to induce granulocyte colony-stimulating factor and stem cell factor, thereby
expanding neutrophil progenitors in the bone marrow and spleen as well as increasing
mature neutrophils in the blood [1-3]. IL-17 also induces ELR+ CXC chemokines, which attract neutrophils [2,3]. By contrast, IFN-γ opposes neutrophil recruitment by downregulating expression of
neutrophil chemoattractants [4]. Analysis of polarized T cell subsets and genetically deficient mice has provided
insight into the distinct effector functions of IL-17 and IFN-γ; however, the interplay
between IL-17 and IFN-γ in vivo remains complex [5,6]. Moreover, downstream effector mechanisms mediating pathological consequences may
be tissue- and pathogen-specific and are largely unresolved. For example, Th17 cell-mediated
protection is critical during bacterial pneumonia [2]. IL-17-mediated neutrophil recruitment to the infection site also indicates a protective
role for Th17 cells during oropharyngeal candidiasis [7]. By contrast, Th17-mediated inhibition of both protective Th1 responses and antimicrobial
neutrophil functions increased tissue destruction following gastric candidiasis and
pulmonary aspergillosis [8]. These differences may reflect distinct infection sites, as indicated by the distinct
immune responses to Candida albicans, which are dependent upon the anatomical site of infection [7].

Viral infections are often dominated by Th1 responses. However, the coemergence of
Th17 and Th1 cells has recently been documented in several infections, including human
immunodeficiency virus [9], simian immunodeficiency virus [10] and cytomegalovirus [11]. A deleterious role of IL-17 is implied by acute lung injury associated with IL-17-mediated
neutrophil recruitment during influenza virus infection [12]. By contrast, Th17 responses are protective against lethal influenza virus infection
in IL-10-deficient mice [13]. Similarly, IFN-γ-mediated protection during herpes simplex virus-1 corneal infection
correlated with reduced IL-17 production and subsequent neutrophil infiltration [14]. However, the function of IL-17 during central nervous system (CNS) viral infections,
including human immunodeficiency virus encephalitis, is unclear, although Th17 cells
promote Theiler’s murine encephalomyelitis virus persistence and chronic demyelination
by limiting the antiviral cytotoxic T-lymphocyte response [15].

In contrast to the limited information on IL-17 function during viral encephalitis,
analysis of experimental autoimmune encephalitis (EAE) has revealed numerous insights
into effector mechanisms as well as crosstalk between Th1 and Th17 cells [16]. Although the inflammatory CNS disease multiple sclerosis and its animal model EAE
were historically associated with a Th1 immune response [17,18], a pro-inflammatory role of IFN-γ was contradicted by substantially increased disease
severity and mortality in mice deficient in IFN-γ (GKO) or the IFN-γR [19,20]. The correlation between increased EAE severity, enhanced Th17 responses and neutrophil
infiltration into the CNS of GKO mice suggested that IFN-γ might be protective by
inhibiting the Th17 response [21]. Although IL-17−/− mice are susceptible to EAE [22], adoptive transfer of polarized encephalitogenic CD4+ T cells support Th17 cells as detrimental participants in EAE [23,24]. However, the pathogenic mechanisms associated with Th17 cells remain an ongoing
challenge and may involve multiple pathways. These include excessive CNS neutrophil
infiltration and release of degrading enzymes, free radicals and pro-inflammatory
cytokines, direct IL-17-mediated neuronal toxicity [25], and/or secretion of granulocyte macrophage colony-stimulating factor (GM-CSF) as
the pathogenic effector molecule [26-28]. These data suggest that the balance between IFN-γ and IL-17 effector functions,
as well as their regulation of neutrophils may dictate the outcome of non autoimmune-driven
CNS inflammation, such as viral encephalitis.

During encephalomyelitis induced by the strain designated JHMV, CD4+ T cells not only contribute to antiviral effects by enhancing CD8+ T cell function within the CNS [29] but also mediate viral control in absence of CD8+ T cells [30]. Nevertheless, they also contribute to both clinical disease and demyelination [30]. To define the role of CD4+ relative to CD8+ T cells in viral encephalitis, memory CD4+ T cells from immunized donors were transferred into infected severe combined immunodeficiency
(SCID) mice [31]. This study revealed an early morbidity and mortality in infected recipients of CD4+ T cells lacking the ability to secrete IFN-γ compared to recipients of IFN-γ-sufficient
CD4+ T cells or infected unreconstituted control mice [31]. Notably, both memory populations were equally effective in controlling virus replication
[31]. The lethal outcome was specific for CD4+ T cells lacking IFN-γ [31], but not for a similar memory CD8+ T cell population deficient in IFN-γ [32]. These data suggest that mortality was related to immune effector functions specific
to CD4+ T cells and controlled by IFN-γ.

In this study, SCID recipients of GKO CD4+ T cells infected with JHMV were characterized by extensive neutrophil accumulation
and IL-17 expression within the CNS. Neutrophil infiltration in the absence of IFN-γ
correlated with significantly elevated levels of CXCL1, independent of IL-17. Moreover,
comparison of infected recipients of wild-type (WT) CD4+ T cells depleted of IFN-γ and recipients of GKO CD4+ T cells depleted of IL-17 revealed mortality was due to IL-17, irrespective of abundant
neutrophil accumulation. IFN-γ introduced by co-transfer of WT CD4+ T cells with IL-17-producing GKO CD4+ T cells abrogated the detrimental effects of IL-17 without affecting IL-17 transcription
within the CNS. These data thus segregate the effects of toxic neutrophil components
from IL-17-mediated pathogenesis.

T cell purification and adoptive transfer

BALB/c Thy1.1 and GKO donors were immunized by intraperitoneal (i.p.) injection with
2 × 106 PFU of JHMV. Donor splenocytes were prepared four to sixteen weeks post immunization.
CD4+ T cells were purified by positive selection using anti-CD4-coated magnetic beads
(Miltenyi Biotec Inc., Auburn, CA, USA). Purity of the purified population was assessed
by flow cytometry using fluorescein isothiocyanate- (FITC) labeled anti-CD4 (clone
GK1.5), phycoerythrin- (PE) labeled anti-CD8 (clone 53-6.7) and peridinin chlorophyll
protein- (PerCP) labeled anti-CD19 (clone 1D3) mAbs (BD Pharmingen, San Diego, CA,
USA). Recipients received 5 × 106 donor CD4+ T cells composed of 100% Thy1.1 (WT), 100% GKO or a 50/50% mixture of Thy1.1/GKO
(WT/GKO) CD4+ T cells by intravenous (i.v.) injection coupled with a single i.p. injection of 250
μg of anti-CD8 mAb (clone TIB.210). Mice were challenged with virus two to three hours
after adoptive transfer. For neutrophil depletion, mice received i.p. injections of
either 500 μg of anti-Ly-6G (clone 1A8) or anti-Gr1 (clone RB6-8C5) mAb every other
day until sacrifice, starting two days before infection. Depletion was confirmed in
both cases by flow cytometric analysis using anti-Ly-6G (clone 1A8) mAb in addition
to examination of hematoxylin and eosin- (H&E) stained sections of brain. Only data
for the anti-Ly6G experiments are shown. No differences in survival relative to control-treated
mice were observed following treatment with either neutrophil-depleting mAb. Similarly,
for anti-IFN-γ treatment, mice received i.p. injections of 500 μg of anti-IFN-γ (clone
XMG1.2) mAb every other day, starting two days before infection. For anti-IL17 treatment,
mice received i.p. injections of 1 mg of anti-IL-17A (clone 1D10) mAb at day zero
and six post infection (p.i.).

Isolation of central nervous system-derived cells

After brain homogenization and centrifugation to obtain supernatants for virus determination
as described above, cell pellets were resuspended in RPMI containing 25 mM HEPES,
pH 7.2 and adjusted to 30% Percoll (GE Healthcare Bio-Sciences BA). A 1 ml underlay
of 70% Percoll was added prior to centrifugation at 800 x g for 30 minutes at 4°C.
Cells were recovered from the 30% to 70% interface and washed with RPMI medium prior
to analysis.

In vitro T cell stimulation

Cytokine expression by CD4+ T cells derived from cervical lymph nodes of SCID recipients were analyzed directly
at day eight p.i. without stimulation with viral antigen. For analysis of cytokine
production by cells prior to transfer, JHMV was adsorbed to donor splenocytes for
60 minutes at 4°C and cells cultured for six days in RPMI complete, 10% FCS at 2.5 × 106 cells/ml. Cytokine production from both splenic cultures or ex vivo lymph node cells was measured following four hours stimulation with PMA (10 ng/ml)
(Acros Organics, Geel, Belgium) and ionomycin (1 μM) (Calbiotech, Spring Valley, CA,
USA). Monensin (2 μM) (Calbiotech) was added to the cultures for the last two hours.
After stimulation, cells were harvested and stained for surface expression of CD4.
Cells were then permeabilized using the cytofix/cytoperm kit (BD Pharmingen) according
to the manufacturer’s instructions and stained for intracellular FITC-IFN-γ and PE-IL-17.

In contrast to memory GKO CD4+ T cells derived from JHMV-immunized donors, memory GKO CD8+ T cells did not trigger early mortality in infected SCID recipients [32]. These data suggest that IFN-γ deficiency was not the sole factor controlling early
death. WT CD4+ T cell recipients were depleted of IFN-γ to confirm that a CD4+ T cell factor distinct from IFN-γ controls disease outcome. The modestly reduced
survival rate of IFN-γ-depleted WT CD4+ T cell recipients (Figure 2A) demonstrated IFN-γ blockade did not reproduce the mortality of GKO CD4+ T cell recipients. The efficiency of IFN-γ blockade within the CNS was confirmed
by analyzing IFN-γ-dependent MHC class II expression on microglia [34]. In contrast to class II expression on the vast majority of microglia in recipients
of WT CD4+ T cells, class II remained undetectable in anti-IFN-γ-treated WT recipients (Figure 2B), confirming inhibition of local IFN-γ signaling within the CNS. IFN-γ depletion
also had minimal effects on T cell recruitment into the CNS, reducing the CD4+ T cells within the inflammatory population from 15.4% to 12.3% (data not shown).
In support of the role of IFN-γ in regulating neutrophils, IFN-γ-depleted WT recipients
exhibited vastly increased CNS neutrophil infiltration, approaching the numbers found
in GKO CD4+ T cell recipients (Figure 2C). In addition to confirming IFN-γ-mediated control of CNS neutrophil recruitment
[4], these data reassert that abundant CNS neutrophils are insufficient to account for
early mortality.

IL-17 mediates mortality, independent of neutrophils

Neither IL-6 nor IL-1β, whose over expression is associated with adverse effects on
the CNS [35,36], were increased in the CNS of GKO compared to WT CD4+ T cell recipients (Figure 3A). Previous data demonstrated that TNF and inducible nitric oxide synthase were also
not associated with early mortality of GKO recipients [31]. Indeed, passive transfer of neutralizing anti-TNF mAb was unable to alter the mortality
of the GKO CD4+ T cell recipients (data not shown). These results suggested additional factor(s)
intrinsic to GKO CD4+ T cells in mediating disease outcome. Inhibition of IL-17 production by IFN-γ [37], suggested IL-17 as a potential candidate. Consistent with this concept, IL-17 mRNA
expression was increased in the CNS of GKO CD4+ T cell recipients (Figure 3B), although IL-17 is not expressed in the CNS of infected WT mice [38]. Importantly, IL-17 mRNA remained below detection not only in SCID-infected control
mice lacking T cells, but also in recipients of WT CD4+ T cells depleted of IFN-γ (Figure 3B), both of which are characterized by vast CNS neutrophil infiltration (Figure 1A). Although neutrophil-derived IL-17 has been implicated in enhancing tissue damage
during reperfusion injury [39], these data suggest that neutrophils recruited into the CNS do not secrete IL-17
during acute viral encephalitis. Expression of IL-17 mRNA only in GKO CD4+ T cell recipients also ruled out a potential contribution of resident CNS cells.
IL-17 expression exclusively in the CNS of GKO recipients thus implied that the source
of IL-17 was the GKO-derived CD4+ T cell population itself. In support of this concept, transcript levels encoding
IL-22, another cytokine produced by Th17 cells [40], were also significantly increased in infected GKO recipients compared to WT recipients
and infected SCID control mice (Figure 3C). By contrast, IL-21, a CD4+ T cell-derived cytokine known to provide helper functions to CD8+ T cells and B cells [41] was expressed at similar levels in both the GKO and WT CD4+ T cell recipient groups (Figure 3C). IL-17 production by CD4+ T cells in the CNS of GKO recipients was confirmed by immunofluorescence histochemistry.
A substantial fraction of T cells within the CNS of GKO recipients expressed IL-17.
Moreover, all IL-17 positive cells co-expressed CD3 (Figure 3D), indicating that T cells are the predominant source of IL-17 within the CNS of
SCID recipients. In contrast to the CNS, only ~8% of T cells in the cervical lymph
nodes of GKO recipients secreted IL-17 at day eight p.i. (Figure 3E), suggesting enrichment of IL-17-expressing T cells within the CNS. To determine
if IL-17 expression is imprinted during the primary response following immunization
of GKO donor mice, cytokine expression was analyzed in the memory WT and GKO T cell
populations prior to transfer (Figure 3F). WT memory CD4+ T cells prominently expressed IFN-γ and very little, if any, IL-17 following in vitro stimulation. By contrast, immunization of GKO mice primed a small fraction of memory
CD4+ T cells capable of producing IL-17. These results were consistent with IFN-γ-mediated
inhibition of Th17 cells [42] and suggested that IL-17 expression was imprinted prior to transfer and re-expressed
in the infected recipients. To confirm a role of IL-17 in the early mortality of GKO
recipients, WT and GKO recipients were treated with anti-IL-17 mAb. Consistent with
the absence of IL-17 mRNA in the CNS of the WT recipients, anti-IL-17 treatment had
no effect on the survival of WT recipients (Figure 4A). By contrast, inhibition of IL-17 in GKO recipients lead to a significant decrease
in mortality, with 73% of mice surviving to day 18 p.i. (Figure 4A). In support of the concept that mortality was not influenced by neutrophils, the
increased neutrophil infiltration in the CNS of GKO recipients was not altered by
anti-IL-17 treatment (Figure 4B), confirming their primary regulation by IFN-γ [4].

Overall these data confirm IFN-γ-mediated control of CNS neutrophil infiltration and
suggested a protective role of IFN-γ during viral encephalitis, via inhibiting IL-17
effector function by either directly reducing Th17 cell expansion and/or CNS entry,
or limiting GM-CSF production. To assess whether GKO CD4+ T cells migrated to the CNS in the presence of WT CD4+ T cells, the relative proportions of each donor population was examined by co-transfer
of Thy1.1 WT CD4+ T cells and Thy1.2 GKO CD4+ T cells. Surprisingly, T cells recruited into the CNS of infected co-transferred
recipients were essentially derived from the GKO memory CD4+ T cells, as less than 20% of CD4+ T cells expressed Thy1.1+ (Figure 6A). Given the large population of infiltrating GKO CD4+ T cells, we next determined if IFN-γ-mediated protection correlated with reduced
IL-17 mRNA expression. Although protective, the minor population of WT CD4+ T that infiltrated the CNS did not reduce expression of IL-17 mRNA in the CNS (Figure 6B). Protection mediated by IFN-γ, despite elevated IL-17, suggested that IFN-γ interferes
with IL-17-mediated signaling events, rather than directly influencing Th17 expression.
This notion was tested by in vitro stimulation of memory CD4+ T cells derived from GKO donors in the presence of recombinant IFN-γ. Exogenous IFN-γ
was indeed unable to downregulate IL-17 production (Figure 6C), supporting the in vivo observation that IFN-γ-expressing WT CD4+ T cells did not alter CNS expression of IL-17 mRNA in WT/GKO recipients (Figure 6B). The maintenance of IL-17 in the presence of IFN-γ in vitro and in vivo indicates that the phenotypes acquired during in vivo primary responses are retained in the transferred memory cells following reactivation
in recipient mice. To confirm this assumption, IFN-γ was depleted in WT/GKO recipients.
WT/GKO recipients treated with anti-IFN-γ succumbed to infection by day nine p.i.
similar to infected recipients of GKO CD4+ T cell (Figure 6D). These data actually suggest that IFN-γ diminishes the detrimental effects of IL-17,
despite the apparent expansion/survival advantage of GKO relative to WT CD4+ T cells in the infected recipients.

Figure 6.IFN-γ-mediated protection prevents IL-17-mediated mortality. (A) Number of CD4+ T cells in the infiltrating population and distribution of Thy1.1 positive cells
measured by flow cytometry at day eight p.i. Data represent means (±SD) of twelve
mice per group combined from three separate experiments. (B) IL-17 mRNA expression determined by quantitative real-time PCR in infected SCID recipients
of WT, WT/GKO or GKO CD4+ T cells. Data represent the mean of two experiments with n = 4 in each group per
experiment. (C) Splenocytes of immunized GKO donors cultured in the presence of JHMV with or without
recombinant IFN-γ (10 ng/ml) for six days and restimulated four hours with PMA/Ionomycin.
Intracellular cytokine expression on CD4+ T cells analyzed by flow cytometry using FITC-IFN-γ, PE-IL-17 and the corresponding
isotype controls. Dot plots are representative of duplicates from two separate experiments.
(D) Survival of infected SCID recipients of WT/GKO (n = 6), WT/GKO + anti-IFN-γ mAb (n = 8)
and GKO (n = 4) CD4+ T cells assessed daily. Data are representative of two separate experiments.

To determine potential mechanisms of IL-17-mediated mortality, IL-17-dependent chemokines
and matrix metalloproteinases (MMPs) [45] were analyzed in JHMV-infected SCID recipients after transfer of WT or GKO CD4+ T cells. Similar expression of CCL2, CCL7 and CCL20 was detected comparing infected
SCID controls and GKO recipients; by contrast CCL2 and CCL7 were upregulated and CCL20
downregulated in recipients of WT CD4+ T cells (Figure 7A). These data suggest that in contrast to EAE, CCL2, CCL7 and CCL20 chemokine expression
is regulated by IFN-γ rather that IL-17 during JHMV infection. Moreover, no significant
difference in CXCL2 mRNA was found comparing SCID-infected controls and recipients
of either WT or GKO CD4+ T cells (Figure 7A), supporting CXCL1 as the major neutrophil chemoattractant during JHMV infection.
CNS infection with JHMV induces a limited number of MMPs, that is, MMP9, MMP3 and
MMP12 [46]. As MMP9 is specifically expressed by neutrophils [47], abundant neutrophil recruitment in the CNS of GKO T cell recipients (whether or
not treated with anti-IL17) correlated with MMP9 expression (Figure 7B). MMP3 and MMP12 mRNA expression were also upregulated in GKO recipients compared
to infected SCID controls and WT recipients, suggesting a potential role of these
MMPs in GKO mortality by mediating tissue destruction (Figure 7B). However, survival of GKO recipients treated with anti-IL17 also expressed increased
MMP3 and MMP12 mRNA (Figure 7B), suggesting that MMP3 and MMP12 play no role in the early mortality of GKO recipients.
Finally, to investigate a potential contribution of GM-CSF to the rapid disease progression,
relative levels of GM-CSF were measured in the CNS of SCID-infected controls, and
recipients of WT and GKO CD4+ T cells. GM-CSF mRNA expression was increased in GKO recipients relative to controls
and WT CD4+ T cell recipients. These data were reminiscent of enhanced GM-CSF expression by Th17
compared to Th1 cells in EAE [27] and suggested a potentially detrimental role during JHMV encephalomyelitis. However,
the increased survival of GKO recipients treated with anti-IL17 mAb did not correlate
with a decrease in GM-CSF expression. These results indicate that GM-CSF expression
correlated with IFN-γ deficiency, but not with an IL-17 mediated feedback loop. Nevertheless,
these data suggest that IFN-γ directly affords protection from mortality by interfering
with detrimental IL-17-mediated events, distinct from those mediating EAE.

Figure 7.Alterations in chemokines, MMPs and GM-CSF mRNA do not correlate with IL-17-mediated
mortality. (A) mRNA expression analyzed in the CNS of naïve mice, infected control mice, and SCID
recipients of WT or GKO CD4+ T cells at day eight p.i. Data represent the mean (±SEM) of four individual mice
per group (B) MMP9, MMP3, MMP12 and GM-CSF mRNA expression measured in the CNS of naïve, controls,
infected SCID recipients of WT, GKO or GKO + anti-IL-17 CD4+ T cells at day eight p.i.. Data are representative of the mean (±SEM) of four individual
mice per group.

Discussion

IFN-γ and IL-17 are major effector molecules of tissue inflammation that play opposing
roles in neutrophil recruitment/accumulation [4,48,49]. While their distinct influence on disease has been demonstrated during autoimmune-mediated
neuroinflammatory responses, the interplay between IL-17 and IFN-γ, specifically the
effects on downstream targets remain controversial. Furthermore, during microbial
infections, protective and detrimental effects of IFN-γ and IL-17 depend on the pathogen
and prominent cell types affecting microbial control [50-52]. The present study evaluated how the absence of IFN-γ secretion by CD4+ T cells contributes to a rapid lethal outcome during viral encephalomyelitis, without
altering viral control. Early virus-induced mortality in SCID recipients of GKO virus-specific
memory CD4+ T cells correlated with both IL-17 production and extensive neutrophil accumulation
in the CNS. Selective blockade of either neutrophils or IL-17 demonstrated that early
mortality did not correlate with CNS neutrophil recruitment, but rather with IL-17.
This was confirmed by the prolonged survival of recipients of anti-IFN-γ mAb-treated
WT recipients, which were characterized by extensive neutrophil inflammation, but
an absence of IL-17.

Neutrophil-independent pathogenic effects of IL-17 in the JHMV model contrast with
non-CNS viral infectious models including the influenza virus and herpes simplex virus-1
infections, which attribute Th17 cell-mediated pathogenesis to neutrophil attraction
[12,14]. However, neutrophil depletion following severe influenza virus infection also suggests
that neutrophils play a protective, rather than a deleterious role [53]. Our data also contrast with the deleterious role of neutrophils during EAE [49,54]. Adoptive transfer of Th17 cells leads to excessive CNS neutrophil migration after
EAE induction, while impaired neutrophil recruitment restrains leukocyte access into
the CNS [49], indicating a prominent role of neutrophils in disrupting the blood-brain barrier.
However, in contrast to EAE, neutrophils are not essential for the loss of blood-brain
barrier integrity following sublethal JHMV infection [55]. By contrast, JHMV-induced encephalomyelitis demonstrates that IFN-γ plays a more
prominent role than IL-17 in regulating CNS neutrophil recruitment and/or retention
by downregulating ELR+ neutrophil chemokine expression. Increased neutrophils correlated with high CXCL1
expression in the CNS of both IFN-γ-depleted WT recipients lacking IL-17, as well
as in GKO recipients treated with anti-IL-17 Ab. Moreover, neutrophil infiltration
was reduced by co-transfer of WT and GKO CD4+ T cells, despite sustained IL-17 expression in the CNS. These results are consistent
with early studies identifying IFN-γ as a critical factor regulating CNS neutrophil
infiltration [4], as well as recent observations implicating IFN-γ as a dominant molecule controlling
CNS inflammation [26].

Despite evidence implicating IL-17 as a pathogenic mediator, independent of neutrophils,
the mechanism(s) involved in IL-17-induced mortality of JHMV-infected mice remain
unclear. Identical viral burden at day eight p.i. in all recipients [31] indicated that IL-17 does not alter control of virus replication, in contrast to
its role in facilitating viral persistence following Theiler’s murine encephalomyelitis
virus infection [15]. Sustained Ag independent interaction between Th17 and neuronal cells during EAE
correlated with increased neuronal damage due to IL-17-mediated neurotoxicity [25]. Increased gray matter infection, especially in neuronal cells, is associated with
premature death following JHMV infection of mice deficient in innate immune components
[56]. In addition, there is a preferential distribution of CD4+ T cells in the gray matter of GKO recipients compared to WT recipients [31], suggesting the possibility that in absence of IFN-γ, IL-17-secreting CD4+ T cells localize proximal to uninfected neurons, contributing to neuronal dysfunction
and premature death. However, few neurons are infected early during JHMV pathogenesis
in SCID mice and the types of infected cells were similar in all groups, suggesting
no alteration in viral tropism [31]. In addition, no differential neuronal loss was found comparing GKO and WT recipients
[31]. Similarly, increased expression of GM-CSF in GKO recipients compared to the WT counterparts
suggested that GM-CSF might also contribute to disease outcome following JHMV infection.
GM-CSF was implicated as a pathogenic effector molecule secreted by Th17 cells during
EAE [27,28]. However, the survival of GKO recipients treated with anti-IL17 did not correlate
with a decrease in GM-CSF expression. Although GM-CSF expression is reduced by IFN-γ
[27], the data do not support a pathogenic role of GM-CSF in early mortality of JHMV-infected
GKO recipients.

IL-17 mRNA expression in GKO CD4+ T cell recipients suggested Th17 cells as the primary mediators of disease. Nevertheless,
IL-17 can also be produced by neutrophils, γδ T cells, NK and CD8+ T cells [57,58]. A deleterious contribution of neutrophil-derived IL-17, suggested during kidney
ischemia-reperfusion [39], was ruled out by the inability of neutrophil-depletion to rescue mice from early
death, as well as the absence of IL-17 mRNA in WT recipients treated with anti-IFN-γ,
despite high CNS neutrophil infiltration. IL-17 production by CD4+ T cells derived from immunized GKO donors prior to transfer supports GKO CD4+ T cells as the primary source of IL-17. Moreover, stimulation of WT donor CD4+ T cells strongly induced IFN-γ, but not IL-17, indicating that virus-specific Th17
cells only differentiate in the absence of IFN-γ. These results support previous observations
of a minor, if any, role of Th17 cells in the pathogenesis of JHMV-infected immunocompetent
WT mice [38] and corroborate the inhibitory function of IFN-γ on Th17 differentiation during T
cell priming [59]. However, our data are novel in demonstrating that memory GKO CD4+ T cells are committed in their ability to produce IL-17 when restimulated in the
recipient host, even in the presence of IFN-γ. Although unanticipated, this finding
was confirmed by the inability of IFN-γ to downregulate IL-17 production in GKO donor
cells in vitro, as well as on in vitro-differentiated mature Th17 cells [15]. Similarly, the IL-27 suppressive function on Th17 differentiation from naïve CD4+ T cells could not be reproduced on memory Th17 cells [60], supporting the stability of committed Th17 cells. Importantly, the prolonged survival
of co-transferred recipients, despite sustained CNS IL-17 expression, suggests that
IFN-γ overcomes the deleterious effects of IL-17. However, the mechanisms by which
IFN-γ overrides IL-17 function remain unclear. In EAE, IL-17 exerts detrimental effects
via signaling in resident CNS cells, with astrocytes implicated as major targets [45]. However, Th17 cell localization proximal to neurons also implicates potential dysregulation
of neuronal function [25]. Responsiveness of both cell types to IFN-γ [61,62] suggests IFN-γ may counteract signaling molecules downstream of the IL-17R.

Conclusions

This study demonstrates that IL-17, in the absence of IFN-γ, can accelerate mortality
during viral encephalomyelitis by a mechanism independent of the magnitude of CNS
neutrophil infiltration and reversible by IFN-γ.

Competing interests

The authors declare they have no competing interests.

Authors’ contributions

CS designed and performed the experiment, collected and analyzed data, and wrote the
manuscript. SAS designed and performed the research, interpreted data and wrote the
manuscript. DRH analyzed and interpreted data. RMR interpreted data. DJC provided
materials, interpreted data and edited the manuscript. CCB designed the research,
provided materials, interpreted data and wrote the manuscript. All authors read and
approved the final manuscript.

Acknowledgments

The authors thank Wenqiang Wei, Kate Stenson and Shabbir Hussain for technical assistance.
This work was supported by National Institutes of Health grant NS18146 and National
Multiple Sclerosis Society fellowship grant FG-1791-A-1 to CS.